An FSHD Antisense Therapy Primer

DNA inside the cell (left) is transcribed into mRNA (blue), which is recognized and bound by an antisense oligonucleotide (red). This blocks the translation of the mRNA into protein. This method can be used to design a therapy that targets the DUX4 gene, which is thought to cause muscle damage in FSH muscular dystrophy.
Image credit: Robinson R. RNAi Therapeutics: How Likely, How Soon? PLoS Biol. 2004;2(1):e28.

Yi-Wen Chen DVM PhD

Q&A With Dr. Yi-Wen Chenby JIM ALBERT, Eldersburg, Maryland

Antisense therapy is a form of treatment for genetic disorders. In the past year antisense drugs have been approved by the FDA for the treatment of two types of muscular dystrophies: some forms of Duchenne muscular dystrophy, and spinal muscular atrophy. While antisense therapy for the potential treatment of FSHD is still in the preclinical stage, we do hear occasional encouraging research results involving antisense technology aimed at FSHD. My interview with Yi-Wen Chen, DVM PhD, of the Children’s National Medical Center and George Washington University presents an interesting primer for a basic understanding of antisense technology and the current state of antisense research in fighting FSHD. Thanks to the generosity of our donors, the FSH Society is funding research, described in this article, by Dr. Chen’s and Dr. Jones’ labs.

Q. What is antisense therapy?
A. Antisense therapy is a form of treatment for genetic disorders. One approach is to create a synthetic molecule that will bind to the messenger RNA (mRNA) produced by the target gene, thus inactivating that gene, or turning if “off.”

Q. How does antisense therapy work?A. If you can recall back to high school biology, there is DNA and RNA, which are nucleic acids that play complementary roles in living cells. DNA is what makes up the chromosomes of the cell. A cell’s genetic information is transferred between DNA and RNA in a process called transcription, where the DNA is used as a template to create a strand of mRNA. In a process called translation, the cell uses the mRNA to create proteins. DNA is contained in the cell nucleus while protein creation via mRNA takes place in the ribosome of the cell with the additional help of ribosomal proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). You can think of the ribosome as the cell’s “protein factory.”

You might remember that DNA is made up of the four nucleobases: adenine (A), cytosine (C), guanine (G), and thymine (T). RNA is made up of the four nucleobases: adenine (A), cytosine (C), guanine (G), and thymine (T), and uracil (U). In RNA, which is important to antisense therapy particularly for FSHD, A binds to U, and C binds to G.

A given “recipe” for a protein is defined by a sequence of nucleobases. For a very simple example, AAGGUC is referred to as the sense sequence for creating a given protein. We want the complementary, binding, or antisense sequence to bind with that sense sequence and turn it off. In our example, that antisense sequence would be UUCCAG. Since mRNA is single stranded, if we can bind to it, we can essentially prevent it from doing its job in the ribosome and stop it from making the target protein.

Q. Aren’t most proteins defined by very long strands of nucleobases?
A. Yes, they are, often a few thousand as is the case with DUX4. However, we’ve found that we only need a small antisense sequence to bind with the mRNA to degrade it and turn it off. One of the antisense oligonucleotide molecules studied in our laboratory is called “third-generation antisense (3GA).” This work is in collaboration with Idera Pharmaceuticals in Boston. Idera found that a 19-length set of nucleobases, referred to as a 19-mer, is most effective in shutting down DUX4. In addition, a unique strategy was used to reduce the immune response in mice injected with the 3GA and improve the efficacy of the antisense therapy. This is one of the challenges of antisense therapy. We want the muscle cells to accept the antisense therapy and not reject it, and in addition increase the half-life of the therapy while limiting toxicity. (Half-life is the amount of time a molecule stays in the body before it gets degraded.)

Q. When we read about antisense therapy, we often see the terms oligonucleotide and Morpholino. Can you explain those terms
A. Oligonucleotide means short DNA or RNA molecules consisting of a small number of nucleotides (A’s, T’s, C’s, and G’s). When we refer to oligonucleotides with regard to antisense therapy, we are referring to antisense oligonucleotides. Antisense oligonucleotides are the single strands of complementary DNA or RNA that prevent or alter protein translation of a target mRNA. To increase stability and reduce toxicity, chemical modifications of the backbones of the antisense oligonucleotides have been developed. Morpholino is one type of antisense oligonucleotide with a specific type of modified backbone. The advantages of Morpholino antisense oligonucleotides are long half-life and reduced toxicity. The disadvantage is that it does not enter muscle cells naturally unless the muscle is “leaky,” as in Duchenne muscular dystrophy.

Q. How is antisense therapy applicable to FSHD?
A. FSHD is believed to be caused by the aberrant expression of the DUX4 gene resulting in the production of DUX4 protein, which is toxic to skeletal muscle. Since antisense therapy can be used to target and remove specific mRNA, the goal is to turn off DUX4 via antisense therapy by degrading the mRNA that is responsible for manufacturing DUX4 protein. If we can do this, we can stop FSHD at what is believed to be the root cause, DUX4 translation, and all subsequent downstream effects of DUX4 would hopefully be relieved.

Q. In theory, could antisense therapy be applicable to both FSHD1 and FSHD2?
A. In theory, yes. Antisense therapy could be applicable to both FSHD1 and FSHD2 since, although there are different genetic mechanisms that define FSHD1 and FSHD2, both result in the aberrant expression of DUX4.

Q. How many different antisense therapies are involved in your research?
A. Idera Pharmaceuticals has tested about 20 different compounds in initial screening studies focusing on DUX4. Of those 20 compounds, five proved effective enough and moved to in vitro and in vivo studies in our lab.

Q. Speaking of in vivo (in living organism) studies, what FSHD animal model are you utilizing in your DUX4 antisense in vivo studies?
A. We’re making use of the FlexD mouse created by Peter Jones, PhD, University of Nevada, Reno School of Medicine. Creating a useful animal model for FSHD has been a challenge to research for quite a while. It’s difficult to create a mouse model that expresses DUX4 without the DUX4 becoming lethal to the mouse. The amount of DUX4 expressed in the FlexD mouse can be controlled via exposure to an enzyme, and the amount of “leaky” DUX4 in the mouse is working out well in our in vivo antisense research.

Q. Does your in vivo DUX4 antisense research involve local or systemic delivery?
A. Both. We are studying delivery of antisense for FSHD via local injection into muscle and systemic delivery via subcutaneous (beneath the skin) injection. We have found subcutaneous injections work well for the antisense therapy to reach muscles, but we may be looking at intravenous delivery as well.

Q. Are there existing FDA approved antisense therapies for other forms of muscular dystrophy?
A. Yes. In the past year the FDA has approved eteplirsen for the treatment of some types of Duchenne muscular dystrophy and SPINRAZA® (nusinersen) for the treatment of spinal muscular atrophy (SMA).

Q. Are you able to learn anything from existing approved muscular dystrophy antisense drugs?
A. There is certainly always something to be learned from previous successes. The strategies developed to increase half-life and reduce toxicity are beneficial to FSHD research. The advancement of delivery methods is critical to developing antisense treatments for FSHD since FSHD patients’ muscles are not as “leaky” as in Duchenne. However, there are differences between the approved antisense therapies for Duchenne and SMA and potential antisense drugs for FSHD. The Duchenne and SMA drugs work to improve a faulty protein in patients. In FSHD, we need to stop the creation of a protein.

Q. The million-dollar question: Do you expect antisense therapy for FSHD to progress to human clinical trials, and if so, how soon might we expect to see that?
A. Based on our current knowledge of antisense oligonucleotide strategies, I am hopeful that antisense therapy for FSHD will progress to human clinical trials soon. The timeline depends on whether we are able to deliver the antisense oligonucleotides systemically and demonstrate efficacy and safety in vivo using animal models. There are several research groups studying different antisense strategies currently, and I believe that we will have promising candidates for clinical trials in the near future.

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9 responses to “An FSHD Antisense Therapy Primer”

Very interesting. Thanks for your hard work. I am an Internal medicine physician and my son is 19 and has early onset FSHD1. Hope and pray things progress well and beyond preclinical. Will trials (as mentioned above at Rochester) with testosterone and GH therapy or any other forms of therapy preclude patients entering antisense trials? Best educated guess on this? Thank you Ron

That’s a good question. The answer is ultimately up to the specific clinical trial investigator, who sets the inclusion criteria for the trial. It’s possible that a study would not want to include patients who are taking a treatment that could potentially skew the results. For example, if a study cohort included some patients who have taken testosterone and rHGH along with some who have not, it might make it difficult to know whether the effects seen after treatment with the study drug are due solely to the study drug or could involve effects of the other testosterone and rHGH treatment. On the other hand, if the study drug is expected to work in a way that is unrelated to any effects that testosterone and rHGH might have, then it may not matter. With regard to gene therapies, it’s possible that being in one gene therapy trial would probably rule out being in any other gene therapy trials. This is a good question to ask when enrolling in any trial.

In addition to everything June stated being correct, it is worth adding that there currently are no antisense drugs in clinical trial. Should such a drug reach clinical trial, it will more than likely follow the standard path of Phase 1 to Phase 2 to Phase 3 with possible sub-phases between each major phase.

Phase 1 clinical trials are almost always conducted on healthy individuals. Any FSHD patients would not be accepted in such a Phase 1 trial.That is not always the case, but it is the norm.

If you were considering participation in the Testosterone/HGH trial in Rochester, I would suggest this article not have a large impact on your decision. I believe the Rochester Tesosterone/HGH trial is currently recruiting which means it likely won’t be long before it begins, if it hasn’t already. Each phase of a clinical trial is usually measured in many months to a year or more with time in between trials to study data and make adjustments before considering moving on to the next phase. There would likely not be any overlap and then going forward if there is any exclusion criteria to a given trial such as if you were taking a testosterone supplement, there is generally a determined wash-out period (usually measured in a few weeks to a month or so) between stopping a treatment and when you can participate in the trial.

Again… these are generalizations I am making on clinical trials in general. Each is unique with its own set of inclusion and exclusion criteria.

As an FSHD patient myself I have some personal experience with FSHD clinical studies and therapeutic clinical trials that I would be happy to share with you. If you contact June directly, she will gladly offer you my email address if there is any way I could help you.

I have familial FSHD. All my relatives on paternal grandmothers side has FSHD. Some are living in India. Some uncles are living beyond 90 years of age. Some are walking some on wheelchairs.
My question to you is if antisense therapy cannot be extradited? Can FDA give a fast track approval since FSHD is a rare genetic disease?
Neela